Has anyone ever done this digestion. The digestion efficiency seems very low. The plasmid is very bright and the insert looks very faint on the gel? There is no dam methylation problem btw. Anyone know what the issue is?
They are both compatible with Buffer 4. I did not try them separately but will. Yeah I did add BSA but only to see if it made a difference and it did not. I normally dont add BSA to any digests and they all work fine.
How big is the insert? Since EB density is correlated with the size of the dsDNA, it is normal to see very dim bands after digestion if you have a small insert to start with.
I wouldn't expect any problem (and had none) using buffer 2 or buffer 4 and Biolab enzymes. However, careful with XbaI as overlapping dam methylation TCTAGAtc methylation prevents digestion. If you know the sequence, check it and regrow plasmid in dam-negative E. coli strain, if needed. Otherwise, one of your enzymes might simply be dead.
Ya, I faced this problem too. Both the restriction enzymes, XbaI and SpeI have the same sticky ends, CTAG. As a result, DNA cut by one enzyme can stick to DNA cut by the other enzyme. However, after ligation, the resulting sequence does not match the recognition site of either XbaI or SpeI. This is called a mixed site. The formation of mixed sites is the basis of BioBrick Standard Assembly where they can introduce new RE sites.
For cloning please don't use XbaI and SpeI together.